Professor Graeme Henderson is Professor of Pharmacology and Head of
Department, Department of Pharmacology, School of Medical Sciences,
University of Bristol, University Walk, Bristol BS8 1TD, UK.

Introduction

Preparations of the opium poppy papaver somniferum have been
used for many hundreds of years to relieve pain. In 1803, Sertürner
isolated a crystalline sample of the main constituent alkaloid, morphine,
which was later shown to be almost entirely responsible for the analgesic
activity of crude opium. The rigid structural and stereochemical
requirements essential for the analgesic actions of morphine and related
opioids led to the theory that they produce their effects by interacting
with a specific receptor.1 The concept that there is more than
one type of opioid receptor arose to explain the dual actions of the
synthetic opioid nalorphine, which antagonises the analgesic effect of
morphine in man but also acts as an analgesic in its own right. Martin
(1967) concluded that the analgesic action of nalorphine is mediated by a
receptor, later called the k-opioid receptor, that is different from the morphine
receptor.2 Evidence for multiple receptors, m, k and s, came from the
demonstration of different profiles of pharmacological activity in the
chronic spinal dog with the prototype agonists morphine, ketazocine and
N-allylnormetazocine (SKF 10047).3 The existence of the
d-receptor
was subsequently proposed to explain the profile of activity in
vitro of the enkephalins (the first endogenous opioid peptides), and
on the basis of the relative potency of the non-selective opioid
antagonist naloxone to reverse endogenous opioid peptide inhibition of the
nerve-evoked contractions of the mouse vas deferens.4 Its
existence was further confirmed by radioligand binding studies using rat
brain homogenates.

It is now clear from work carried out in many laboratories over the
last 20 years that there are 3 well-defined or "classical" types of opioid
receptor µ, d and k. Genes encoding for these receptors have been cloned.5, 6, 7,
8 More recently, cDNA encoding an "orphan" receptor was identified
which has a high degree of homology to the "classical" opioid receptors;
on structural grounds this receptor is an opioid receptor and has been
named ORL1 (opioid receptor-like).9 As would be
predicted from their known abilities to couple through pertussis
toxin-sensitive G-proteins, all of the cloned opioid receptors possess the
same general structure of an extracellular N-terminal region, seven
transmembrane domains and intracellular C-terminal tail structure. There
is pharmacological evidence for subtypes of each receptor and other types
of novel, less well-characterised opioid receptors, e, l, i, z, have also been
postulated. The s-receptor, however, is no longer regarded as an opioid
receptor.

Receptor Subtypes

m-Receptor
subtypesThe MOR-1 gene, encoding
for one form of the m-receptor, shows approximately 50-70% homology to the
genes encoding for the d-(DOR-1), k-(KOR-1) and orphan (ORL1) receptors. Two splice
variants of the MOR-1 gene have been cloned, differing only in the
presence or absence of 8 amino acids in the C-terminal tail. The splice
variants exhibit differences in their rate of onset and recovery from
agonist-induced internalization but their pharmacology does not appear to
differ in ligand binding assays.10 Furthermore, in the MOR-1
knockout mouse, morphine does not induce antinociception demonstrating
that at least in this species morphine’s analgesia is not mediated through
d- or
k-receptors.11 Similarly morphine did not exhibit
positive reinforcing properties or an ability to induce physical
dependence in the absence of the MOR-1 gene.

m1 and m2:The
m1/m2 subdivision was proposed by Pasternak and
colleagues to explain their observations, made in radioligand binding
studies, that [3H]-labelled-m, -d and -k ligands displayed biphasic binding
characteristics.12 Each radioligand appeared to bind to the
same very high affinity site (m1) as well as to the
appropriate high affinity site (m, d or k) depending on the radioligand
used. Naloxazone and naloxonazine were reported to abolish the binding of
each radioligand to the m1-site. Furthermore, in in vivo
studies it was observed that naloxazone selectively blocked
morphine-induced antinociception but did not block morphine-induced
respiratory depression or the induction of morphine dependence.13,
14 Subsequent work in other laboratories has failed to confirm this
classification.

Is there another, novel form of the
m-opioid
receptor?Several related
observations suggest the existence of a novel form of m-receptor at which analogues of morphine with substitutions
at the 6 position (e.g. morphine-6b-glucuronide, heroin and 6-acetyl morphine) are
agonists, but with which morphine itself does not interact.15
In antinociception tests on mice it has been reported that morphine does
not exhibit cross tolerance with morphine-6b-glucuronide, heroin or 6-acetyl
morphine. Furthermore, in mice of the CXBX strain morphine is a poor
antinociceptive agent whereas morphine-6b-glucuronide, heroin and 6-acetyl
morphine are all potently antinociceptive. The 6-substituted morphine
analogues do not appear to be acting through d- or k-receptors because the
antinociception they induce is not blocked by selective d- or k-receptor antagonists,
whereas 3-methoxynaltrexone has been reported to antagonise
morphine-6b-glucuronide- and heroin-induced antinociception without affecting
that induced by morphine, [D-Pen2, D-Pen5]enkephalin
(DPDPE , d-selective) or U50488 (k-selective).16

Recently it has been reported that heroin and morphine-6-glucuronide,
but not morphine, still produce antinociception in MOR-1 knockout mice in
which the disruption in the MOR-1 gene was engineered in
exon-1.17 The same authors observed that in other MOR-1
knockout mice in which exon-2, not exon-1, had been disrupted, all three
agonists were ineffective as antinociceptive agents. They conclude that
the antinociceptive actions of heroin and morphine-6-glucuronide in the
exon-1 MOR-1 mutant mice are mediated through a receptor produced from an
alternative transcript of the MOR-1 gene differing from the MOR-1 gene
product, the m-opioid receptor, in the exon-1 region. To substantiate this
conclusion they report that in RT-PCR experiments using primers spanning
exons 2 and 3, a MOR-1 gene product was still detected in MOR-1 knockout
mice.

d-Receptor
subtypesThe DOR-1 gene is the only
d-receptor gene cloned to date. However,
two, overlapping subdivisions of d-receptor have been proposed (d1/d2
and dcx/dncx) on the basis of in vivo and in
vitro pharmacological experiments.

d1 and d2:The
subdivision of the d-receptor into d1 and d2 subtypes
was proposed primarily on the basis of in vivo pharmacological
studies (Table 1). In rodents in vivo, the supraspinal
antinociceptive activity of DPDPE can be selectively antagonised by
7-benzylidenenaltrexone (BNTX) or [D-Ala2,
D-Leu5]enkephalyl-Cys (DALCE)18, 19 whereas the
antinociceptive activity of [D-Ala2]-deltorphin II (deltorphin
II) and [D-Ser2, Leu5]enkephalyl–Thr (DSLET) can be
reversed by naltriben or naltrindole 5¢-isothiocyanate (5¢-NTII).18, 19,
20 Furthermore, while mice develop tolerance to the antinociceptive
effects of repeated injections of either DPDPE or deltorphin II, this
tolerance appears to be homologous in that there is no cross tolerance
between these ligands.21In vivo,d1- and
d2-receptor-induced antinociception can be differentially
antagonised by blockers of different types of potassium
channels.22

Table 1 . Putative ligands for d-receptor
subtypes

Receptorsubtype

Antagonists

Competitive

Nonequilibrium

d1

DPDPE /
DADLE

BNTX

DALCE

d2

Deltorphin II
/ DSLET

Naltriben

5¢-NTII

N.B. DPDPE may not in fact be a selective
d1 agonist but may also be a partial agonist at
d2-sites.23

The best evidence from in vitro
experiments to support thed1 and d2 subdivision of
d-receptorscomes from inhibition of adenylyl cyclase
activity in membranes from rat brain24, 25 and from the
d-receptor-mediated elevations of intracellular Ca2+ in
the ND8-47 cell line26 where BNTX selectively antagonised
DPDPE, and naltriben selectively antagonised deltorphin II. Surprisingly,
little selectivity was seen in radioligand displacement
studies.24 The converse has been observed in studies on
neuronal cell lines. Two distinct d-receptor binding sites were
observed in radioligand binding experiments on SK-N-BE cells.27
Studies on NG108-15 cells28 or the human neuroblastoma cell
line, SH-SY5Y,29, 30 have failed to find any functional
evidence for d-receptor subtypes.

The pharmacological properties of the cloned
DOR-1 receptor are somewhere between those predicted for either the
d1 or d2 subtypes. DPDPE and deltorphin II are both
potent displacers of [3H]-diprenorphine binding to mouse and
human recombinant receptors, which is not consistent with either the
d1 or d2 classifications.31 In contrast,
[3H]-diprenorphine binding to the mouse recombinant receptor is
more potently displaced by naltriben than BNTX, suggesting that the cloned
receptor is of the d2 subtype. It will be of importance to
determine in the DOR-1 knockout mouse if analgesia can still be induced by
either d1- or d2-receptor selective agonists.

dcx and dncx: The
dcx and dncx subdivision of d-receptors was based on the
hypothesis that one type of d-receptor (dcx) was complexed with m-receptors (and perhaps
also k-receptors) whereas the other type of d-receptor (dncx) was not associated
with an opioid receptor complex.32 It was originally observed
that sub-antinociceptive doses of agonists at the dcx receptor (e.g. low
doses of DPDPE), potentiated m-receptor-mediated analgesia, an effect which could be
antagonised by 5¢-NTII. On the other hand, at higher doses, DPDPE then acted as an
agonist at the dncx-receptor and itself induced analgesia which was
reversed by DALCE. Data obtained from subsequent radioligand binding
studies have been interpreted as demonstrating the existence of further
subtypes of the dncx receptor i.e. d(ncx-1) and d(ncx-2).
More recently it has been suggested that the d(ncx-1) receptor is in
fact synonymous with the d1-receptor and the dcx-receptor synonymous
with the d2-receptor of the previous
classification.33

k-Receptor subtypesThe situation
regarding the proposals for subtypes of the k-receptor is rather more complex than for the m- and d-receptors, perhaps because of the continuing use of
non-selective ligands to define the putative sites. The evidence for the
need for sub-division of the k-receptor
comes almost entirely from radioligand binding assays.

The first characterisation of a k-receptor binding site in brain
came from work using [3H]-ethylketocyclazocine
(EKC).34 Crucial to this success was the use of the guinea-pig
brain where k-sites are present in relative abundance, and of "suppression", or
quenching of the binding of this non-selective ligand to m- and d-sites, by incubation
with non-radioactive ligands that bound selectively at these other
sites.

Studies of [3H]-EKC binding in guinea-pig spinal cord
pointed to the existence of a non-homogeneous population of high-affinity
binding sites, and led to the first proposal for k1- and k2-sites
distinguished by their sensitivity to DADLE.35 The
DADLE-sensitive k2 site bound b-endorphin with high affinity, and
was later identified with the recognition site of the e-receptor in
brain.36 Another study using [3H]-EKC identified a
k-site in
bovine adrenal medulla, with a pharmacology similar to that of the
k1-site in guinea-pig cord37 but labelling
with [3H]-etorphine revealed two additional sites, one
resembling k2 that bound [Met]enkephalyl-Arg-Gly-Leu with high
affinity and another termed "k3" or "MRF" that bound
[Met5]enkephalyl-Arg-Phe with high affinity.

The k1/k2 terminology has more recently been applied
by other groups to the putative subtypes defined in other tissues in their
hands, but it is not always clear how closely the common nomenclature
reflects a common pharmacology. The introduction of the first selective
k-agonist
U-50,488 and its congeners (U-69,593, PD 117302, CI 977, ICI 197067)
led to a refinement of the definition of the putative subtypes, but
pointed to the need for careful considerations of the effect of technical
differences in assays and of species as a possible explanation for
discrepancies. Thus a direct comparison of the binding of
[3H]-EKC in guinea-pig and rat (with suppression of binding to
m- and
d-sites)
pointed to the existence of a high affinity k1-site that predominated
in guinea-pig brain and was selectively sensitive to U-69,593, and a low
affinity, U-69,593-insensitive k2-site that predominated
in rat brain.38 Others resorted to the binding of
[3H]-bremazocine to reveal U-69,593-insensitive k2-binding
sites; in contrast to the k2-site originally defined in guinea-pig
spinal cord, the k2-site in brain after suppression of k1 was
insensitive to DADLE.39

Subdivision of the k1-site in guinea-pig brain into k1a and
k1b, was proposed to resolve the complex displacement of
either [3H]-EKC or [3H]-U-69,593 with dynorphin B
and a-neo-endorphin which both preferentially bound to the proposed
k1b sub-subtype.40 The same study proposed the
existence of a k3 subtype, insensitive to U-50,488, that was identified
from the binding of [3H]-naloxone benzoylhydrazone. The
pharmacology of this later "k3-site" is rather different from the
k3/MRF site of bovine adrenal medulla, and has been
proposed to be the receptor mediating the antinociceptive effect of
nalorphine, Martin’s "N"-receptor.41

Nomenclature differences appear to have arisen in the context of
subtyping of the k1-subtype. Using binding surface analyses to allow
highly accurate estimation of binding parameters, the binding of
[3H]-U-69,593 resolved two binding sites termed k1a and
k1b. The ligand demonstrating the highest affinity, and
around 30-fold preference, for the "k1a binding site" was
a-neo-endorphin.42 More recently putative k1a- and
k1b-sites in mouse brain were identified from complex
displacement curves against the binding of [3H]-U-69,593, in an
attempt to compare the pharmacology of the mouse k1-sites, with that at
the cloned rat KOR stably expressed in a host neuroblastoma cell
line.43 Based on the high affinity of bremazocine and
a-neo-endorphin, it was deemed "consistent to term the cloned KOR a
k1b subtype".

Rothman (1990) also reported subdivision of the k2-binding of
[3H]-bremazocine into 2a- and 2b-sub-subtypes.42 The
k2b-site had high affinity for b-endorphin and DADLE, reminiscent
of the original k2-binding site of guinea-pig spinal cord. The
k2a- and k2b-sites in guinea-pig brain have undergone a
further subdivision (sub-sub-subtypes?) on the basis of investigations
using a combination of depletion (of m- and d-sites) and suppression, against
the binding of 6b-[125I]-3,14-dihydroxy-17-cyclopropylmethyl-4,5a-epoxymorphinan
([125I]OXY).44 So were defined the k2a-1 and
k2a-2 sites, having relatively high and low affinities
respectively for nor-BNI and enadoline (CI-977), and k2b-1 and
k2b-2 sites with high and low affinities for DAMGO and
a-neo-endorphin.

Definitive functional pharmacological evidence supporting the existence
of this confusing number of putative subtypes of the k-receptor is lacking,
because of the absence of subtype-specific antagonists. It has been
reported however, that pretreatment with the isothiocyanate analogue of
U-50,488 called (-)-UPHIT, was able to produce a long-lasting block of the
antinociceptive effect of U-69,593 in the mouse without affecting the
action of bremazocine, while treatment with the non-selective antagonist
WIN 44,441 (quadazocine) blocked selectively the antinociception with
bremazocine.45,46 These findings provide obvious support for
the k1-k2 subdivision; the pharmacological corollary
is that (-)-UPHIT and WIN 44,441 are antagonists with selectivity for the
k1-and k2-subtypes respectively, at least in the
mouse.

Correlating genes with m-, d- and k-receptor subtypesAlthough there is as yet
little evidence for different genes encoding the different subtypes of
m-, d- and
k-receptor these subtypes may result from
different post-translational modifications of the gene product
(glycosylation, palmytoylation, phosphorylation, etc), from receptor
dimerization to form homomeric47 and heteromeric
complexes,48, 49, 50 or from interaction of the gene product
with associated proteins such as RAMPs.51

The Orphan Receptor

Extending the screening of genomic and cDNA libraries, perhaps in an
effort to identify putative subtypes of the classical opioid receptors,
resulted in the identification of a novel receptor that bore as high a
degree of homology towards the classical opioid receptor types, as they
shared among each other. The receptor was identified in three species:
rat, mouse and man, with the degree of homology among the species variants
more than 90%. Although the putative receptor has had as many names as the
number of groups who reported its identification,52 there is
some consensus for the use of the original designation for the human form,
"ORL1". Workers in the field are, however, divided in their
preferred terminology for the endogenous peptide agonist for
ORL1 with both "nociceptin"53 or "orphanin
FQ"54 being used with roughly equal frequency.

Although the ORL1 receptor was accepted as a member of the
"family" of opioid receptors on the basis of its structural homology
towards the classical types, there is no corresponding pharmacological
homology. Even non-selective ligands that exhibit uniformly high affinity
towards m-,
k- and
d-receptors, have very low affinity for the ORL1
receptor, and for this reason as much as for the initial absence of an
endogenous ligand, the receptor was called an "orphan opioid receptor".
Close comparison of the deduced amino-acid sequences of the four receptors
highlights structural differences that may explain the pharmacological
anomaly. Thus there are sites near the top of each of the trans-membrane
regions, that are conserved in the m-, k- and d-receptors, but are altered in
ORL1. Work with site-directed mutants of ORL1 (rat)
has shown that it is possible to confer appreciable affinity on the
non-selective benzomorphan bremazocine by changing Ala213 in
TM5 to the conserved Lys of m, k and d, or by changing the Val-Gln-Val276-278
sequence of TM6 to the conserved Ile-His-Ile motif.55

A splice variant of the ORL1 receptor from rat has been
reported ("XOR")56 with a long form (XOR1L) containing an
additional 28 amino acids in the third extracellular loop. In the
homologous receptor from mouse (also sometimes referred to as "KOR-3")
five splice variants have been reported to date.57

ORL1-Receptor subtypesSelective high affinity
ligands with which to attempt pharmacological definitions of the
ORL1 receptor are few in number (Table 2). Besides the natural
heptadecapeptide agonist nociceptin/orphanin FQ and some closely related
peptides, the only other ligands offering high affinity and selectivity
belong to a class of peptides obtained by a positional scanning approach
to combinatorial libraries of hexapeptides.58 Being basic
peptides highly susceptible to degradation, all of those agents are chancy
tools in the hands of the unwary. So the paucity of safe and sure
pharmacological tools may partly explain some of the confusion in the
literature regarding the effect of nociceptin in tests of response latency
to noxious stimulation; antinociception, pro-nociception/hyperalgesia,
allodynia, or no overt effect, have all been reported.

Table 2. Selective opioid ligands

Receptor type

µ-Receptor

d-Receptor

k-Receptor

ORL1

Selective agonists

endomorphin-1endomorphin-2DAMGO

[D-Ala2]-deltorphin I[D-Ala2]-deltorphin
IIDPDPESNC
80

enadolineU-50488U-69593

nociceptin /
OFQAc-RYYRWK-NH2*

Selective
antagonists

CTAP

naltrindoleTIPP-yICI 174864

nor-binaltorphimine

None as yet**

Radioligands

[3H]-DAMGO

[3H]-naltrindole[3H]-pCI-DPDPE[3H]-SNC
121

[3H]-enadoline[3H]-U69593

[3H]-nociceptin

*Related combinatorial library hits are also
selective agonists.58 **Ac-RYYRIK-NH2
has been proposed to be an ORL1 antagonist61 whereas
the putative antagonist [Phe1y(CH2-NH)Gly2]nociceptin(1-13)NH259
appears to be a partial agonist.

Although the results of some studies have been interpreted as pointing
to the existence of subtypes of ORL1, this conclusion is so far
premature in most cases. The most reliable pharmacological definition of
receptors is based on differences in antagonist affinity, and in this
context the absence of useful antagonists for ORL1 is
particularly galling to pharmacologists. Although the synthetic analogue
of the N-terminal tridecapeptide of nociceptin, [Phe1y(CH2-NH)Gly2]nociceptin(1-13)NH2
was first reported to be a selective antagonist,59 increased
use of this peptide points to it having agonist actions. There are no
grounds for saying that this peptide is an antagonist at ORL1
receptors in the periphery, but an agonist in the brain (not least because
agonist actions in the periphery, and antagonist actions in the brain have
been reported) and that these differences in efficacy point to
differences in the receptors. Although differences in the affinity
for [Phe1y(CH2-NH)Gly2]nociceptin(1-13)NH2
may be found between central and peripheral sites,60 and there
may indeed be different "subtypes" of ORL1 in the brain and
periphery, the safest conclusion for the moment is just that
[Phe1y(CH2-NH)Gly2]nociceptin(1-13)NH2
is a partial agonist, and that the observed differences in efficacy are
consistent with differences in receptor reserve.

Very recently a peptide related to the combinatorial hexapeptide
library hit acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2
(Ac-RYYRWK-NH2;Table 2), but with isoleucine
substituting for tryptophan, was reported to block the effects of
nociceptin/orphanin FQ in rat cortex (stimulation of GTPg35S binding) or heart (positive
chronotropic effect in isolated myocytes). Although this peptide, like all
of its structural homologues, was originally reported to be a potent
agonist, but with somewhat less than full efficacy,58 it will
be important to see if the antagonist activity of
Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (Ac-RYYRIK-NH2 ) at
the ORL1 receptor61 is
confirmed.

Less Well-Characterised Opioid Receptors

In addition to the µ-, d-, k- and
ORL1-receptors, several other types of
opioid receptor have been postulated. Since the contractions of the
isolated vas deferens of the rat are much more sensitive to inhibition by
b-endorphin
than by other opioid peptides, it was suggested that this tissue contains
a novel type of opioid receptor, the e-receptor, that is specific for
b-endorphin.62 The rabbit ileum has been proposed to
possess i-receptors, for which the enkephalins have high affinity but which
are distinct from d-receptors.63 A very labile l-binding site with high affinity
for 4,5 epoxymorphinans has been found in freshly-prepared rat membrane
fragments64 and there is evidence that opioids inhibit growth in S20Y
murine blastoma cells by an action at yet another receptor type called the
z-receptor.65 The e-, l-, i-and z-receptors are poorly characterised
and wider acceptance of their existence awaits further experimental
evidence, in particular isolation of their cDNAs.

Although originally classified as such, the s-receptor appears not to be an opioid receptor but rather
the target for another class of abused drugs, phencyclidine (PCP) and its
analogues.66 Phencyclidine is an effective blocker of the ion
channel associated with the N-methyl-D-aspartate (NMDA) receptor where it
binds to the same site as MK 801.67

Endogenous Ligands

In mammals the endogenous opioid peptides are mainly derived from four
precursors: pro-opiomelanocortin, pro-enkephalin, pro-dynorphin and
pro-nociceptin/orphanin FQ.68, 69, 70, 53 Nociceptin/orphanin
FQ is processed from pro-nociceptin/orphanin FQ and is the endogenous
ligand for the ORL1-receptor; it has
little affinity for the µ-, d- and k-receptors.53, 54 The amino acid sequence of
nociceptin/orphanin FQ has homology with other opioid peptides especially
the prodynorphin fragment dynorphin A (Table 3), suggesting a close
evolutionary relationship between the precursors. Nociceptin/orphanin FQ,
however, has a C-terminal phenylalanine (F) whereas peptides derived from
the other precursors all have the pentapeptide sequence
TyrGlyGlyPheMet/Leu (YGGFM/L) at their N-termini. These peptidesvary in their affinity for µ, d- and k-receptors, and have negligible affinity for
ORL1-receptors, but none binds
exclusively to one opioid receptor type.71, 53b-endorphin is
equiactive at µ-and d-receptors with much lower affinity for k-receptors; the
post-translational product, N-acetyl-b-endorphin, has very low affinity
for any of the opioid receptors.72, 73 [Met]- and
[Leu]enkephalin have high affinities for d-receptors, ten-fold lower
affinities for µ-receptors and negligible affinity for k-receptors.71 Other products of processing of
pro-enkephalin, which are N-terminal extensions of [Met]enkephalin, have a
decreased preference for the d-receptor with some products, e.g. metorphamide
displaying highest affinity for the µ-receptor.71 The opioid
fragments of pro-dynorphin, particularly dynorphin A and dynorphin B, have
high affinity for k-receptors but also have significant affinity for µ- and
d-receptors.71

Endomorphin-1 and endomorphin-2 are putative products of an as yet
unidentified precursor, that have been proposed to be the endogenous
ligands for the µ-receptor where they are highly selective.74
The endomorphins are amidated tetrapeptides and are structurally unrelated
to the other endogenous opioid peptides (Table 3). Although the study of
the cellular localisation of these peptides is at an early stage,
endomorphin-2 is found in discrete regions of rat brain, some of which are
known to contain high concentrations of m-receptors.75
Endomorphin–2 is also present in primary sensory neurones and the dorsal
horn of the spinal cord where it could function to modulate nociceptive
input.76

In comparison to the mainly non-selective mammalian opioid peptides
(the exceptions being the endomorphins), amphibian skin contains two
families of D-amino acid-containing peptides that are selective for µ- or
d-receptors. Dermorphin is a
µ-selective heptapeptide Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 without significant affinity at d- and k-receptors.77 In contrast, the
deltorphins - deltorphin (dermenkephalin;
Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2),
[D-Ala2]-deltorphin I and [D-Ala2]-deltorphin II
(Tyr-D-Ala-Phe-Xaa-Val-Val-Gly-NH2,
where Xaa is Asp or Glu respectively) - are highly selective for
d-opioid
receptors.78

Table 3. Mammalian endogenous opioid
ligands

Precursor

Endogenous peptide

Amino acid
sequence

Pro-opiomelanocortin

b-Endorphin

YGGFMTSEKSQTPLVTL-FKNAIIKNAYKKGE

Pro-enkephalin

[Met]enkephalin[Leu]enkephalin

Metorphamide

YGGFMYGGFLYGGFMRFYGGFMRGLYGGFMRRV-NH2

Pro-dynorphin

Dynorphin ADynorphin
A(1-8)Dynorphin Ba-neoendorphinb-neoendorphin

YGGFLRRIRPKLKWDNQYGGFLRRIYGGFLRRQFKVVTYGGFLRKYPKYGGFLRKYP

Pro-nociceptin /
OFQ

Nociceptin

FGGFTGARKSARKLANQ

Pro-endomorphin*

Endomorphin-1Endomorphin-2

YPWF-NH2YPFF-NH2

*Presumed to exist, awaiting discovery

Effector Mechanisms

The opioid receptor family, in common with the somatostatin receptor
family, is somewhat unusual in that all of the cloned opioid receptor
types belong to the Gi/Go-coupled superfamily of
receptors. Opioid receptors do not couple directly with Gs or
Gq and none of the cloned receptors forms a ligand-gated ion
channel. It was originally thought that m- and d-receptors coupled through Gi/Go proteins to activate an inwardly rectifying potassium
conductance and to inhibit voltage-operated calcium conductances whereas
k-receptors
only inhibit voltage-operated calcium conductances. However it is now
known that the k-receptor is, in
some cell types, alsocoupled to activation of an inwardly
rectifying potassium conductance.79 It seems highly likely,
therefore, that all ofthe opioid receptors will share common
effector mechanisms. Indeed, many papers have recently appeared
demonstrating that the ORL1-receptor
couples to the same effector systems as the other more extensively studied
opioid receptors. It should be borne in mind that, given the heterogeneity
of ai, ao, b and g subunits
which may combine to form a trimeric G protein, there may well be some
subtle differences in the downstream effector mechanisms to which opioid
receptors are coupled if one type of opioid receptor is unable to interact
with a certain form of Gi/Go heterotrimer. However,
different responses evoked in different cell types in response to
activation of different opioid receptors or even in response to activation
of the same receptor are likely to reflect changes in the expression of G
proteins and effector systems between cell types rather than any inherent
differences in the properties of the receptors themselves.

Opioid receptor activation produces a wide array of cellular responses
(Table 4). Although the pertussis toxin sensitivity has not been assessed
in all instances it is highly likely that in each the first step is
activation of Gi or Go . The functional significance
of many of these opioid receptor-mediated effects is still unclear, but
two recent observations on changes in neurotransmitter release following
acute and chronic exposure to opioids are worthy of special mention
because they provide potential solutions to long-asked questions.

The periaqueductal grey region (PAG) is a major
anatomical locus for opioid activation of descending inhibitory pathways
to the spinal cord and is thus an important site for m-receptor-induced analgesia. Opioids do not excite
descending fibres directly but disinhibit them by inhibiting spontaneous
GABA release from local GABAergic interneurones.80 This
inhibition of transmitter release results from activation of a
dendrotoxin-sensitive, voltage-sensitive potassium conductance. The
mechanism by which the voltage-sensitive potassium conductance is
activated appears to be through activation of phospholipase
A2 (PLA2) with subsequent
metabolism of arachidonic acid along the 12¢-lipoxygenase pathway because the inhibition of GABA
release can be inhibited by quinacrine and 4-bromo-phenacylbromide,
inhibitors of PLA2, and by baicalein,
an inhibitor of 12¢-lipoxygenase.
This proposed mechanism of opioid action also explains the synergy between
opioids and non-steroidal analgesic drugs (NSAIDs) in producing analgesia
because in the presence of a NSAID, with the cyclo-oxygenase enzymes
inhibited, more of the arachidonic acid produced by opioid activation of
PLA2 can be diverted down the
12¢-lipoxygenase pathway.

The cellular locus of opiate withdrawal has long been the Holy Grail of
opioid biologists. Over 20 years ago, it was shown that following chronic
exposure of NG108-15 neuroblastoma x glioma hybrid cells to opiates,
withdrawal resulted in a rebound increase in adenylyl
cyclase;81 the functional significance of this observation for
opiate withdrawal in brain neurones has remained obscure. Recently,
Williams and colleagues82 have observed an increase in the
release of the inhibitory neurotransmitter GABA, in the nucleus accumbens
during opiate withdrawal. This effect could be mimicked by the adenylyl
cyclase activator, forskolin, and inhibited by protein kinase A
inhibitors. Therefore, as proposed over 25 years ago by the late Harry
Collier, rebound adenylyl cyclase activity in withdrawal may be the
fundamental step in eliciting the withdrawal behaviour.83

Development and Clinical Applications of Opioid Ligands

Among the receptors for the many neuropeptides that exist in the
nervous system, the opioid receptors are unique in that there existed
before the discovery of the natural agonists, an abundance of non-peptide
ligands with which the pharmacology of the receptors was already defined.
In current terms relating to the drug-discovery process, we would consider
the 4,5-epoxy-methylmorphinan opioid alkaloids morphine, codeine and
thebaine as "natural-product hits" on which were based chemical programmes
to design analogues with improved pharmacology (Figure 1). The effects of
morphine to reduce sensitivity to pain or to inhibit intestinal motility
and secretion, have continued to be exploited clinically, however the
presence of other undesirable effects (e.g. depression of respiration,
tolerance/dependence, effects on mood) provided the stimulus to seek
analogues that were selective in producing analgesia. Thus a
semi-synthetic di-acetylated analogue of morphine was introduced in the
19th century in the mistaken belief that this compound (heroin) had those
desired properties. More radical changes to the morphinan nucleus were
subsequently explored in various synthetic programmes, in many early cases
resulting in the development of low efficacy partial agonists.

With the benefit of hindsight, it is possible to conceive an evolution
of those opioid analogues, with a progressive simplification of chemical
structure from the epoxymorphinans (nalorphine, nalbuphine) through the
morphinans such as levorphanol, and the benzomorphans such as pentazocine,
to the phenyl-piperidines including pethidine and the
4-anilino-piperidines as exemplified by fentanyl (Figure 1). The ultimate
simplification of the morphine structure was in the methadone class, with
methadone itself and d-propoxyphene (Darvon). Although thebaine is
virtually inactive, the compound itself was an important chemical
precursor in the synthesis of 14-hydroxy derivatives of morphine, most
particularly the antagonists naloxone and naltrexone. Also derived from
thebaine were the oripavine derivatives, and here the trend of chemical
"simplification" was reversed with the introduction of an additional
six-membered ring that appeared to enhance biological potency. For
example, etorphine is about one thousand times more potent than morphine
as an analgesic, but its use is limited to veterinary medicine as a
sedative for large animals.

For the most part, such compounds have highest affinity for the
m-receptor,
and to a greater or lesser extent produce the full panoply of effects,
good and bad, obtained with morphine. Depending on the level of affinity
and efficacy, such compounds have been used acutely or chronically, to
provide analgesia in cases of mild, through moderate to severe pain, alone
or with adjuncts. The piperidines related to fentanyl include the most
potent non-peptide m-agonists known, and are generally used
peri-operatively, often for the induction and maintenance of anaesthesia.
The use of many of the benzomorphans (as had been found with the first of
the "duallists" nalorphine) has been associated with dysphoric and
psychotomimetic effects in man, a property originally thought to be
attributable to affinity at the non-opioid s-site.

The attractiveness of the prospect for development of selective
k-agonists
as analgesics was based on the preclinical pharmacology in animals of the
6,7-benzomorphans such as ketazocine and its derivatives (Figure 1).
Although those agents are not selective in terms of affinity, their
utility as pharmacological tools is based on their functional selectivity
for the k-receptor, where their efficacy is high. Such agents
produced a powerful antinociceptive effect, but did not substitute for
morphine in dependent animals. A full biochemical and pharmacological
characterisation of the k-receptor was not possible until the discovery of highly
selective agonists in the aryl-acetamides that appear unrelated
structurally to any of the morphine derivatives. The first compound of
this class was U-50,488, but its importance was also as a chemical lead
for the attempted design of related compounds of greater selectivity and
potency. At least two such compounds have entered clinical trials as
centrally acting analgesics, Spiradoline (U-62,066) and enadoline
(CI-977). Although CNS-mediated, mechanism-related side effects of
sedation and dysphoria may limit the potential for development of such
compounds, the prospects for analogues with limited brain penetration to
produce a peripherally mediated analgesic effect in inflammatory
conditions is under exploration, with at least one compound (asimadoline,
EMD-61753) in clinical trials for osteoarthritis. The observation of
neuroprotective properties of k-agonists in pre-clinical models of
cerebral ischaemia has lead to consideration of the possible clinical
development of selective k-agonists for stroke or traumatic head injury. In this
context the sedative properties of k-agonists, and even perhaps their
characteristic diuretic action, may be advantageous.

The discovery of the enkephalins and of the d-receptor, led to the idea that the
peptides themselves might be taken as "leads" for the synthesis of a new
class of opioid agonist that lacked the addictive properties of morphine.
Although such synthetic activities produced many useful experimental
tools, no direct benefit in the form of a drug appeared, in spite of the
attempted development of several enkephalin analogues. It did become clear
from the work of a number of laboratories that activation of the
d-receptor
is associated with antinociception in animals, and the development of a
selective non-peptide agonist is under consideration by a number of
commercial drug houses. In some cases the synthetic strategy is based
directly on structural considerations of the first non-peptide with
significant selectivity, the 6,7-indole analogue of naltrexone,
naltrindole.84 Applying the "message-address" concept that
produced the antagonist naltrindole to a novel series of
octahydroisoquinoline derivatives has been successful in producing
non-peptide d-selective agonists
TAN-6785 or SB 213698.86 Similar considerations do
not serve to explain the existence of another series of novel piperazine
derivatives d-agonists, BW
373U8687 or SNC 80.88 Preclinical studies suggest
that d-agonists may have a superior
profile as analgesics, but this will only be established when such an
agent is successfully introduced into clinical investigation; other
possible applications of selective ligands for this receptor may emerge
from clinical experience.

The prospects for clinical utilities of agonists or antagonists for the
ORL1 receptor can only be the subject of speculation.
Elucidation of the role of the nociceptin/ORL-receptor system in pain
control (and in other areas, for the peptide and its receptor have a dense
and wide investment in the nervous system) must await the initial results
of the drug-discovery process. Only with the availability of non-peptide
selective agonists, and perhaps more particularly antagonists, will it be
possible to undertake the definitive pre-clinical studies that will serve
for the identification of possible clinical targets. There is some
agreement that activation of the ORL1 receptor in the brain
leads to a motor impairment, so it may be that the development of
ORL1 agonists would be
difficult.